Nuclear receptors (NRs) are a major target of drug discovery. NRs are ligand-dependent transcription factors that possess the ability to directly interact with DNA regulating the transcriptional activity of their target genes. These receptors play essential roles in development, cellular homeostasis and metabolism, and they have been implicated in a wide range of diseases and, as such, have been the focus of drug development efforts for the pharmaceutical industry.
In the newest nomenclature for nuclear receptors, the subfamily 1 C (NR1C) comprises three subtypes of mammals Perixome Proliferator Activated Receptors (PPARs): PPARα (also called NR1C1), PPARβ/δ (also called NR1C2) and PPARγ (also called PPARg, glitazone receptor or NR1C3). PPARs control the expression of networks of genes involved in adipogenesis, lipid metabolism, inflammation and maintenance of metabolic homeostasis [Barish et al., 2006]. PPARs activate gene transcription by binding to elements of DNA sequences, known as peroxisome proliferator response elements (PPRE) in the regulatory region of PPAR target genes [Poulsen et al., 2012]. In addition, PPARs negatively regulate the transcription of inflammatory response genes by antagonizing the Activator Protein-1 (AP-1), Nuclear Factor-kappa B (NF-kB), signal transducer and activator of transcription 3 (STAT3) and Nuclear Factor of Activated T-cells (NFAT) signaling pathways [Vanden Berghe et al. 2003].
Among PPARs, PPARg is of special interest because it is involved in the regulation of adipocyte formation, insulin sensitivity and inflammation [Fievet et al. 2006] [Stienstra et al. 2007] [Tontonoz and Spiegelman, 2008]. PPARg is expressed in a range of tissues including adipose tissue, skeletal muscle cells, osteoclasts, osteoblasts, immune cells, and in the central and peripheral nervous system. It is clear that PPARg is the dominant or “master” regulator of adipogenesis, due to the fact that is both sufficient and necessary for fat cell differentiation. The regulatory regions of a large number of genes that play important roles in lipogenesis and insulin sensitivity such as aP2, LPL, adiponectin, and Glut4 contain binding sites for PPARg [Rosen and MacDougald, 2006]. Therefore, activation of PPARg in adipose tissue impacts whole-body insulin sensitivity.
In addition to its role in metabolic homeostasis regulation, emerging effects of PPARg have been reported including anti-inflammatory, anti-tumor and anti-fibrotic potentials especially [Zhao et al., 2006]. TGFb/Smad signaling blockage by PPARg activation leads to decreased collagen deposition in hepatic, pulmonary, and renal fibrosis [Ferguson et al., 2009] [Wang et al., 2007] [Zhang et al., 2009]. On the other hand, activation of PPARg exerts anti-inflammatory activities in several cell types by inhibiting the expression of pro-inflammatory genes, thereby reducing the production of cytokines, metalloproteases and acute-phase proteins [Tontonoz and Spiegelman, 2008]. It also acts increasing anti-inflammatory cytokines, and inhibiting inducible nitric oxide synthase (iNOS) expression [Széles et al., 2007]. Interestingly, PPARg agonists have shown anti-inflammatory and neuroprotective effects in several experimental models of Parkinson's diseases, amyotrophic lateral sclerosis, multiple sclerosis and stroke, as well as in a few clinical studies [Bernardo and Minghetti, 2008]. In this sense it has been shown that PPARg is highly expressed in retinoic acid treated neuronal precursors (NP) and it is involved in two stages of neural differentiation of mouse embryonic stem cells, during and post-NPs formation [Ghoochani et al., 2012]. Additionally, PPARg must formally be considered a tumor suppressor gene in the genetic sense. It is expressed in a variety of tumor cells, and the activation of PPARg by ligands led to either inhibition of cell proliferation or induction of apoptosis [Tachibana et al., 2008] [Tontonoz and Spiegelman, 2008].
The beneficial effects of PPARg activation by specific ligand agonists can be used for the treatment of several chronic diseases such as diabetes, atherosclerosis, rheumatoid arthritis, liver fibrosis, inflammatory bowel diseases, nephropathy, psoriasis, skin wound healing, scleroderma (SSc) neurodegenerative and neuroinflammatory disorders, and cancer.
Among activators of PPARg ligands, the thiazolidindiones (TZDs) are of most clinical importance [Lehmann et al., 1995]. For this reason rosiglitazone and pioglitazone have been largely used so far in the clinical practice. They provide similar effects on glycemic control, as well as a range of similar adverse effects, such as weight gain, fluid retention, and increased risk of hearth failure, which seem to be PPARg mediated. Indeed, rosiglitazone was recently withdrawn in Europe and its use has been restricted in USA as a consequence of increased risk of cardiovascular events in type 2 diabetic patients.
Although TZDs are potent PPARg full agonists (PPARg-fa) their mechanism-based side effects have limited the full therapeutic potential of those compounds [Gelman et al., 2007] [Ciudin et al., 2012]. But the physiologic and therapeutic relevance of the PPARg pathway have promoted new studies to develop newer classes of molecules that reduce or eliminate adverse effects [Ahmadian et al., 2013]. Therefore, much progress has been achieved in the discovery and development of selective PPARg modulators (PPARg-m) as safer alternatives to PPARg-fa. The preclinical and clinical findings clearly suggest that selective PPARg-m have the potential to become the next generation of PPARg agonists: effective insulin sensitizers with a superior safety profile to that of PPARg-fa. [Doshi et al. 2010].
In this sense natural and synthetic cannabinoids are considered PPARg-m that alleviates inflammatory process through activation of PPARg. Some examples of cannabinoid-based PPARg-m are ajulemic acid [Liu et al., 2003], [Burstein S. 2005], WIN55212-2 [Sun and Bennett, 2007], 9Δ-THC and CBD [O'Sullivan 2007], and CBG [Granja et al., 2012].
Some cannabinoid quinone derivatives such as CBD-Q (HU-311, also named VCE-004 in the present invention) and CBG-Q (VCE-003) have been described [Kogan et al., 2004] [Granja et al., 2012]. Interestingly, VCE-004 (also known as HU-331) showed an EC50 of 5 μM, thus presenting four times higher binding affinity than its parent molecule CBD (EC50 of 21 μM), and VCE-003 showed a significantly enhanced binding affinity for PPARg (EC50 2.2 μM) compared to its parent molecule CBG (EC50 12.7 μM) [Granja et al., 2012]. Other CBD quinones such as CBD-1,4-dihydroxyquinone, 4 methyl-CBD-quinone and 4-formyl-methoxy-CBD-quinone have been also described and showed higher affinity for PPARg compared to its parent molecule CDB [WO2011117429 A1]. However the synthesis of those compounds it is very difficult to reproduce and the compounds are very unstable making them impossible for pharmaceutical development.
Quinones represent a class of toxicological intermediates, which can create a variety of hazardous effects in vivo, including acute cytotoxicity and immunotoxicity [Bolton et al., 2000]. The mechanisms by which quinones cause these effects can be quite complex. Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS) that can cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA [Monks and Jones, 2012]. Although there are numerous examples of quinone-based compounds with therapeutic use, the concerns over non-specific toxicity and lack of selectivity, the Michael acceptor motif is rarely introduced by design in drug leads.
The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to endogenous and exogenous stresses caused by reactive oxygen species (ROS) and electrophiles. The key signaling proteins within the pathway are the transcription nuclear factor (erythroid-derived 2)-like 2 (Nrf2) that binds together with small Maf proteins to the antioxidant response element (ARE) in the regulatory regions of target genes. Under basal conditions Nrf2 is retained in the cytoplasm by the inhibitor Keap1 (Kelch ECH associating protein 1). When cells are exposed to oxidative stress, electrophiles, or chemopreventive agents, Nrf2 escapes Keap1-mediated repression and activates antioxidant responsive element (ARE)-dependent gene expression to maintain cellular redox homeostasis [Na and Surh, 2013].
Nrf2 can protect cells and tissues from a variety of toxicants and carcinogens by increasing the expression of a number of cytoprotective genes. Just as Nrf2 protects normal cells, studies have shown that Nrf2 may also protect cancer cells from chemotherapeutic agents and facilitate cancer progression [Na and Surh 2013]. Cancer cells survive persistent endogenous oxygen-mediated stress and become resistant to certain anticancer agents that exert cytotoxicity through ROS production. Under such conditions, an active Nrf2 pathway could maintain a favorable redox balance in cancer cells by keeping ROS levels within a range that promotes their growth and survival. Sustained accumulation or activation of Nrf2 is speculated to confer on a subset of premalignant or cancerous cells an advantageous environment to proliferate, evade apoptosis, metastasize, and tolerate therapeutic intervention.
Inhibition of Nrf2 overexpression has been known to reverse the phenotypic characteristics of cancer cells, lending support to this supposition [Sporn and Liby, 2012]. Constitutive overactivation of Nrf2 has been observed in numerous types of malignancies, such as squamous cell carcinomas, lung cancer, breast cancer, gallbladder cancer, prostate cancer, renal cancer, ependymomas, ovarian epithelial carcinoma, endometrial cancer, and pancreatic cancer [Na and Surh, 2013]. Cancer patients with a constitutively elevated level of Nrf2 expression in their tumor, in general, show a lower survival rate [Solis et al., 2010]. Therefore, Nrf2 activation is considered a prognostic molecular marker for determining the status of cancer progression and contributes to both intrinsic and acquired chemoresistance. Thus, this antioxidant transcription factor may also act as a proto-oncogene and enhanced Nrf2 activity promotes formation and chemoresistance of solid cancers [Sporn and Liby, 2012].
To improve just PPARg agonistic activity, but without inducing activation of Nrf2 in order to avoid potential side effects, present invention has developed a library of novel compounds starting from VCE-004 and Cannabidiol acid (CBDA) as templates and surprisingly it has been found CBD-quinone derivatives (CBD-Q derivatives) with specific modifications in position 3 resulted on novel compounds with high PPARg agonistic effect but lacking electrophilic (Nrf2 activation) and cytotoxic activities. Therefore, the novel compounds are suitable for treating chronic diseases responsive to PPARg modulation.
VCE-004 (compound I), precursor of the CBD-Q derivatives II to X of present invention is an agonistic PPARg ligand that also activates the transcription factor Nrf2, a cellular sensor of oxidative/electrophilic stress reflecting the generation of ROS in VCE-004-treated cells. Therefore chronic treatment with this type of CBD-Q derivatives that activate the Nrf2 pathway may result in tumor promotion, as explained above. In addition, chromenopyrazolediones, which are structural analogues of CBD-Q, induce cytotoxicity in prostate cancer cells through induction of reactive oxigen species (ROS) and PPARg-dependent mechanisms [Morales et al., 2013]. Thus, oxidation of CBD molecule results in a class of CBD-Q compounds such as VCE-004 that activate PPARg and also induce ROS-mediated Nrf2 activation.
Those CBD-Q derivatives of present invention are different from the compounds described by Kogan et al. [Kogan et al., 2004] and Morales et al. [Morales et al., 2013] since the modifications in position 3 confers to the compounds of the present invention the capacity to activate to PPARg and to protect from glutamate-induced cytotoxicity without activating Nrf2. Moreover, CBD-Q derivatives with modifications in position 3 also inhibited TGFb-induced collagen gene transcription and collagen expression. The compounds described in the present invention are also different from the compounds described in WO20011117429, which are unstable, difficult to synthesize and never tested for Nrf2 activation. The CBD-Q derivatives described in the present invention also shown a remarkable low cytotoxicity in cell lines of neuronal origin compared to VCE-004 (compound I) comprised in the state of the art.